Preferential activation of HIF-2α adaptive signalling in neuronal-like cells in response to acute hypoxia.

Stroke causes severe neuronal damage as disrupted cerebral blood flow starves neurons of oxygen and glucose. The hypoxia inducible factors (HIF-1α and HIF-2α) orchestrate oxygen homeostasis and regulate specific aspects of hypoxic adaptation. Here we show the importance of HIF-2α dependant signalling in neuronal adaptation to hypoxic insult. PC12 and NT2 cells were differentiated into neuronal-like cells using NGF and retinoic acid, and exposed to acute hypoxia (1% O2). Gene and protein expression was analysed by qPCR and immunoblotting and the neuronal-like phenotype was examined. PC12 and NT2 differentiation promoted neurite extension and expression of neuronal markers, NSE and KCC2. Induction of HIF-1α mRNA or protein was not detected in hypoxic neuronal-like cells, however marked induction of HIF-2α mRNA and protein expression was observed. Induction of HIF-1α target genes was also not detected in response to acute hypoxia, however significant induction of HIF-2α transcriptional targets was clearly evident. Furthermore, hypoxic insult dramatically reduced both neurite number and length, and attenuated expression of neuronal markers, NSE and KCC2. This correlated with an increase in expression of the neural progenitor and stem cell-like markers, CD44 and vimentin, suggesting HIF-2α molecular mechanisms could potentially promote regression of neuronal-like cells to a stem-like state and trigger neuronal recovery following ischaemic insult. Our findings suggest the HIF-2α pathway predominates over HIF-1α signalling in neuronal-like cells following acute hypoxia.

Chloride co-transporters as possible therapeutic targets for stroke.

Stroke is one of the major causes of death and disability worldwide. The major type of stroke is an ischaemic one, which is caused by a blockage that interrupts blood flow to the brain. There are currently very few pharmacological strategies to reduce the damage and social burden triggered by this pathology. The harm caused by the interruption of blood flow to the brain unfolds in the subsequent hours and days, so it is critical to identify new therapeutic targets that could reduce neuronal death associated with the spread of the damage. Here, we review some of the key molecular mechanisms involved in the progression of neuronal death, focusing on some new and promising studies. In particular, we focus on the potential of the chloride co-transporter (CCC) family of proteins, mediators of the GABAergic response, both during the early and later stages of stroke, to promote neuroprotection and recovery. Different studies of CCCs during the chronic and recovery phases post-stroke reveal the importance of timing when considering CCCs as potential neuroprotective and/or neuromodulator targets. The molecular regulatory mechanisms of the two main neuronal CCCs, NKCC1 and KCC2, are further discussed as an indirect approach for promoting neuroprotection and neurorehabilitation following an ischaemic insult. Finally, we mention the likely importance of combining different strategies in order to achieve more effective therapies.

GABA(A) receptor chloride channels are involved in the neuroprotective role of GABA following oxygen and glucose deprivation in the rat cerebral cortex but not in the hippocampus.

Assays on "ex vivo" sections of rat hippocampus and rat cerebral cortex, subjected to oxygen and glucose deprivation (OGD) and a three-hour reperfusion-like (RL) recovery, were performed in the presence of either GABA or the GABA(A) receptor binding site antagonist, bicuculline. Lactate dehydrogenase (LDH) and propidium iodide were used to quantify cell mortality. We also measured, using real-time quantitative polymerase chain reaction (qPCR), the early transcriptional response of a number of genes of the glutamatergic and GABAergic systems. Specifically, glial pre- and post-synaptic glutamatergic transporters (namely GLAST1a, EAAC-1, GLT-1 and VGLUT1), three GABAA receptor subunits (α1, ß2 and γ2), and the GABAergic presynaptic marker, glutamic acid decarboxylase (GAD65), were studied. Mortality assays revealed that GABAA receptor chloride channels play an important role in the neuroprotective effect of GABA in the cerebral cortex, but have a much smaller effect in the hippocampus. We also found that GABA reverses the OGD-dependent decrease in GABA(A) receptor transcript levels, as well as mRNA levels of the membrane and vesicular glutamate transporter genes. Based on the markers used, we conclude that OGD results in differential responses in the GABAergic presynaptic and postsynaptic systems.

The lack of associations between alleles at the hypoxia-inducible factor 1A C1772T loci and responses to acute hypoxia.

OBJECTIVE: The aim of this study was to investigate the associations between alleles of the hypoxia-inducible factor 1A (HIF1A) C1772T polymorphism and several physiological responses to hypoxia, including the hypoxic ventilatory response (HVR), and serum erythropoietin (EPO), arterial oxygen saturation (Sao2), and acute mountain sickness (AMS) responses during 8 hours of exposure to normobaric hypoxia. METHODS: A total of 76 males participated in the study; 52 participants completed an 8-hour exposure to 12.7% oxygen, during which time Sao2, EPO concentrations, and AMS scores were measured, while 62 individuals took part in an HVR trial (in total 38 individuals completed both protocols). DNA was obtained from leukocytes, and a 346-bp fragment of the HIF1A gene containing the C1772T polymorphism was amplified using polymerase chain reaction. Fragments were sequenced to reveal individual genotypes, and the associations between HIF1A genotype and EPO, Sao2, AMS responses to hypoxia and HVR were examined. RESULTS: The magnitude of the hypoxic responses was highly variable between individuals. The increase in participants' EPO responses ranged from 89% to 388% of baseline values following hypoxia, while Sao2 values during the exposure ranged from 71% to 89%. The HVR ranged from -0.04 to +2.18 L x min(-1) x Sao2 %(-1) among participants. No significant differences in EPO, Sao2, AMS, or HVR results were observed between the HIF1A CC genotype and the combined CT/TT genotype group. CONCLUSION: In this study, the HIF1A C1772T polymorphism does not appear to influence EPO, Sao2, or AMS responses during acute hypoxic exposure, or the magnitude of the HVR.

Consequences of the evolution of the GABA(A) receptor gene family.

1. This paper reviews the evolution of the family of genes present in mammals and other vertebrates that encode gamma-aminobutyric acid (GABA) type A (GABA(A)) receptors, which are the major inhibitory neurotransmitter receptors in the central nervous system (CNS). In mammals, 16 different polypeptides (alpha1-alpha6, beta1-beta3, gamma1-gamma3, delta, epsilon, pi, and theta) have been identified, using recombinant DNA techniques, each of which is encoded by a distinct gene. The products of these genes assemble in diverse combinations to form a variety of receptor subtypes that have different sensitivities to a number of clinically relevant compounds, such as the benzodiazepines (BZs). 2. Based on a number of chromosomal mapping techniques, the majority of the GABA(A) receptor genes have been localized, in man, in four clusters on chromosomes 4, 5, 15, and the X. Furthermore, the genes that are present within these clusters have a conserved transcriptional orientation. It has, therefore, been proposed that the clusters arose largely as a consequence of two whole-genome doublings that occurred during chordate evolution, and that the ancestral cluster contained an "alpha-like," a "beta-like," and a "gamma-like" subunit gene. 3. Our laboratory has identified two additional GABA(A) receptor polypeptides (the beta4 and gamma4 subunits) in a number of vertebrate species; these do not appear to be present in mammals. We discuss here the relationship of the corresponding genes to other GABA(A) receptor genes, and conclude that their products are orthologous to the mammalian theta and epsilon subunits, respectively. 4. The GABA(A) receptor has a number of binding sites for compounds such as BZs, barbiturates, neurosteroids, and certain volatile anaesthetics. However, the only site at which endogenous compounds are thought to be active is the steroid site; this binds steroids such as certain metabolites of progesterone and deoxycorticosterone, which are synthesized in the periphery and CNS. Since the in vivo functional relevance, if any, of binding sites for other classes of compounds (such as the BZs) is unknown, the significance of differences in primary sequence, between different receptor subunits, is uncertain. We suggest that a possibly more important consequence of gene duplication is that it permitted greater flexibility in the level, pattern and regulation of expression of GABA(A) receptor genes.

Differential Localization of Two Alternatively Spliced GABAA Receptor gamma2-Subunit mRNAs in the Chick Brain.

Two variants of the GABAA receptor gamma2 subunit are known to exist, which differ by the presence (gamma2L) or absence (gamma2S) of eight amino acids in the presumed intracellular loop between the third and fourth membrane-spanning domains. These variants have been shown to be generated by alternative splicing of the gamma2-subunit primary gene transcript in mouse (Kofuji et al., J. Neurochem., 56, 713 - 715, 1991), and in bovine and human (Whiting et al., Proc. Natl. Acad. Sci. USA, 87, 9966 - 9970, 1990) brain. We describe here the cloning, from chick (Gallus domesticus) brain, of cDNAs that encode the gamma2L and gamma2S subunits, and report on the regional and cellular localization of the corresponding mRNAs as revealed by in situ hybridization histochemistry with transcript-specific oligonucleotide probes. While the two transcripts are found to be colocalized throughout the chick neuroaxis, certain nuclei (for example, the nucleus isthmi, pars magnocellularis, the nucleus isthmi, pars parvocellularis, the nucleus solitarius and the paleostriatum primitivum) are found to contain predominantly either the gamma2S- or the gamma2L-subunit mRNA. We conclude that receptors that contain either the gamma2S or the gamma2L subunit occur, and that these probably have functionally different roles in the modulation of neurotransmission in the central nervous system. In addition, our data indicate that certain cells may produce both transcripts. Consequently, these will have either a single receptor subtype that contains both a gamma2S and a gamma2L subunit, or two receptor subtypes, one of which contains a gamma2S subunit and the other a gamma2L subunit.